- 學位論文
利用酵母雙雜交技術找尋在大鼠腦中與ClC-2氯離子通道有交互作用的蛋白質
Application of yeast two-hybrid screen to identify ClC-2-interacting proteins in rat brain
摘要
ClC-2是屬於ClC 氯離子通道和運輸器家族的成員之一。ClC-2通道主要位在細胞膜上，並廣泛的表現在各個組織及細胞中，在大腦以及上皮組織中表現量更為豐富。先前的研究指出，ClC-2通道在神經細胞中與調節其神經興奮性有關，人類中若在ClC-2基因有突變可能會導致全身性的癲癇發作。目前對於ClC-2通道在生理上所扮演的角色雖然已有初步的了解，但它在細胞中是如何被調控及表現的，至今尚不清楚。因此為了更進一步了解調控ClC-2通道的分子機制，我們使用酵母雙雜交技術尋找與ClC-2通道相互作用之蛋白質。藉由這些蛋白質在細胞中已知參與的機制、訊息傳導路徑，可以進一步推測C lC-2的調控性質。 我們將ClC-2未在細胞質內的C端CBS1到CBS2 domain之前的這段序列，亦即第581-794個胺基酸接在pGilda載體上，找尋與這段序列有相互作用的蛋白質。對於大鼠腦部cDNA library進行篩選後，共發現了124個蛋白質可能與ClC-2有相互作用。在經過進一步的序列分析，扣除胺基酸序列有frame shift，剩下53個可能與ClC-2有相互作用的蛋白質。目前從中挑選10個蛋白質，進行進一步的實驗以確認其與ClC-2通道的相互作用能力，我們使用X-gal測試分析和leucine需求分析實驗，結果顯示出這10個蛋白質都能在進行X-gal分析時使菌落由白變藍，並且在缺乏leucine的培養皿上生長。接下來我們以共同免疫沈澱法設法確認其相互作用關係。雖然結果顯示NSF (N-ethylmaleimide sensitive fusion protein)、SMAD 1及Carhsp1(calcium regulated heat stable phosphoprotein 1)似乎有被共同免疫沈澱的跡象。但由於訊號太微弱，我們仍不能確認其彼此之間的相互作用關係。在GSTpull down的結果中，NSF似乎能被GST-ClC-2給pull-down。在進一步確認這些蛋白質與ClC-2的相互關係後，我們將利用電生理技術研究這些蛋白質是否會改變ClC-2之電生理特性，以釐清兩者之間的相關性。
並列摘要
ClC-2 is a member of the ClC family of chloride channels and transporters. It is mainly located on the plasma membrane and expressed in various kind of tissues, and most abundant in the brain and epithelium. Previous studies have shown that ClC-2 channel expression in neurons may modulate their membrane excitability. A disruption in the ClC-2 gene was reported to correlate with the presence of generalized epilepsy in human. In spite of the recent progress in the understanding of the physiological and pathophysiological significance of ClC-2 channels, their regulatory as well as signaling pathways remain unclear. Therefore, we applied yeast two-hybrid screen to search for ClC-2-interacting proteins, which will provide important insight on the physiological function of ClC-2 channels. We focused on a sequence between the CBS1-CBS2 region (amino acids 581-794) at the C-terminal tail of ClC-2, which was cloned into the bait vector pGilda. After screening a rat brain cDNA library, 124 prey clones were identified. By eliminating the clones with incorrect reading frames, we have obtained 51 positive clones. 10 potential candidates from 51 positive clones were chosen for further characterization. X-gal assays and Leucine requirement tests were performed to reconfirm the interaction between ClC-2 and potential candidate proteins. All of the 10 candidate proteins showed blue patches on X-gal-containing plates and were grown on leucine-deficient plates, suggesting that these clones may indeed interact with ClC-2 channels. We performed co-immunoprecipitation and GST pull-down assay to verify the interaction between ClC-2 and potential candidates of ClC-2-interacting proteins. Despite of the fact that the signal intensity of co-immunoprecipitation was not optimal, our current data suggest that ClC-2 probably display direct interaction with with NSF (N-ethylmaleimide sensitive fusion protein), SMAD 1, and Carhsp 1 (calcium regulated heat stable phosphoprotein 1). Moreover, our preliminary GST pull-down results also support a direct interaction between NSF and ClC-2. Further experiments, hower, will be required to verify the foregoing observation. In the future, we plan to apply electrophysiological techniques to determine whether these candidate proteins may affect the biophysical properties and/or trafficking process of ClC-2 channels.
參考文獻
延伸閱讀
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